Genomics provides a powerful tool to study questions at the interface of development and evolution. The genital imaginal disc of Drosophila, from which the external genitalia and analia develop, offers a unique opportunity to investigate both how transcriptional networks direct gene expression to specify large-scale developmental changes, and how the expression of these genes has evolved to produce distinct morphology among different species. The goal of this proposal is to understand how the sex determination hierarchy regulates the development and evolution of sex-specific genital morphology. First, whole-genome microarray expression assays of male and female Drosophila melanogaster genital discs will be performed to identify genes regulated by the doublesex branch of the sex hierarchy that are differentially expressed between the sexes. Expression assays using mutants that lack analia and genitalia will be used to further delineate which candidate genes specify analia and genitalia, respectively. Candidate genes identified from these expression assays will be validated molecularly to verify their sex-specificity and direct regulation by Doublesex. Second, the set of male genitalia-specific genes identified in the previous experiment will be used to investigate expression differences underlying the most rapidly evolving external morphological structure in Drosophila: the posterior lobe of the male genital arch. This will be accomplished by assaying the expression of these genes in genital discs of two closely related species, D. simulans and D. mauritiana, which bear striking differences in genital arch shape and size. Pure species gene expression will be compared to gene expression in hybrids between these two species, which bear intermediate genital arch morphology. The set of genes that are misexpressed in the hybrids will be characterized functionally using standard molecular genetic techniques in D. melanogaster. This experiment will allow for the identification of genes important for genital arch development and reveal on how their expression has changed between species. The study of gene regulation in Drosophila is widely applicable to the study of gene regulation in other species, including humans and their primate relatives. The proposed work is relevant to public health because the information learned from this study can be applied to understanding how defects in gene regulation translate into human diseases.